Process for production of human growth hormone using Bacillus Brevis

ABSTRACT

A host-vector system suitable for production of human growth hormone (hGH) and a process for production of hGH using the same are provided. As an hGH expression plasmid, a recombinant DNA wherein a DNA coding for hGH is linked to the 3&#39;-terminal of a DNA containing a promoter region derived from Bacillus brevis is provided, and as a host, especially a mutant Bacillus brevis substantially not exhibiting protease activity to hGH is provided. A microorganism obtained by transforming said host with said hGH expression plasmid efficiently produces hGH when it is cultured.

This application is a 371 of PCT/JP94/00269, filed Feb. 22, 1994.

1. Field of Art

The present invention relates to a process for production of humangrowth hormone (hereinafter sometimes referred to as "hGH") by Bacillusbrevis carrying a DNA coding for hGH as a heterologous gene, as well assaid DNA and Bacillus brevis carrying said DNA.

2. Background Art

The hGH comprises 191 amino acids, and the amino acid sequence thereofis already known (Bancroft, F. C., Expl. Cell Res., 79, 275-278 (1973),or David V. Goeddel et al., Nature, 281, 544-548 (1979)). Since thissubstance has growth-stimulating action, action in fat-metabolism oraction in sugar-metabolism, for a long time it has been used as atherapeutic agent for dwarfism, and recently it has been noticed as atherapeutic agent for senile dementia.

So far, much recombinant DNA research has been carried out using E.coli, and many heterogeneous proteins have been produced in E. coli.However, in this method, since the heterologous protein produced isintracellularly accumulated, not only are extraction of a desiredproduct from the cells and the purification of the desired product fromthe extract time- and labor-consuming, but also it is not easy torecover the desired product in its native form.

On the other hand, for a long time, microorganisms belonging to thegenus Bacillus have been industrially used as producers for variousextracellular enzymes. Among these extracellular enzymes, α-amylase geneof Bacillus amyloliguefaciens) (I. Palva et al., Gene, 22, 229 (1983)),penicillinase gene of Bacillus licheniformis) (C. Chang et al.,Molecular Cloning and Gene Regulation in Bacilli, Academic Press, 659(1982)), and α-amylase gene of Bacillus subtilis (H. Yamazaki et al., J.Bacteriol., 156, 327 (1983) have been cloned, and production andsecretion of heterogeneous proteins using promoters or signal peptidesof the above genes have been reported.

For the production and secretion of heterogeneous proteins by the genusBacillus, Bacillus subtilis is mainly used as a host. However, sincethis microorganism produces a large amount of extracellular proteases,there is a tendency for heterogeneous proteins secreted by a recombinantDNA technique to be degraded and the amount accumulated is notablydecreased.

On the contrary, Udaka et al. succeeded to produce and secrete α-amylase(Japanese Unexamined Patent Publication (Kokai) No. 62-201,583;, H.Yamagata et al., J. Bacteriol., 169, 1239 (1987)), and swine pepsinogen(Shigezo Udaka, the abstracts of the 1987 Meeting of the AgriculturalChemical Society of Japan, p 837 to p 838; Norihiro Tsukagoshi, NipponNogei Kagaku Kaishi, 61, 68 (1987), using a promoter and signal peptideof MWP (Middle Wall Protein) (H. Yamagata et al., J. Bacteriol., 169,1239 (1987); Norihiro Tsukagoshi, Nippon Nogei Kagaku Kaishi, 61, 68(1987)), which is a major extracellular protein of the above-mentionedmicroorganism, using as a host Bacillus brevis 47 which substantiallydoes not extracellularly produce proteases (Japanese Unexamined PatentPublication (Kokai) Nos. 60-58,074, and 62-201,583; FERMP-7224).

In addition, Udaka et al. succeeded in isolating Bacillus brevis HPD31(FERMBP-1087) producing no detectable extracellular protease, and inusing it as a host so as to produce thermostable α-amylase (JapaneseUnexamined Patent Publication (Kokai) No. 63-56,277; the abstracts ofthe 1987 Agricultural Chemical Society of Japan), and human EGF (H.Yamagata et al., Proc. Natl. Acad. Sci. USA, 86, 3589 (1989); JapaneseUnexamined Patent Publication (Kokai) No. 2-31,682).

The production of hGH using as a host E. coli (David V. Geoddel et al.,Nature, 281, 544-548 (1979), Chang C. N. et al., Gene, 55, 189-196(1987)), Bacillus subtilis (M. Honjo et al., J. Biotech., 6, 191-204(1987)), yeast (R. Hiramatsu et al., Appl. Environ. Microbiol., 57,2052-2056 (1991)) or animal cells (Lupker J. H. et al., Gene, 24,281-287 (1983)) has been reported.

DISCLOSURE OF THE INVENTION

As described above, although it has been attempted to produce hGH usingas a host E. coli, yeast or animal cells, all of these procedures arenot satisfactory due to the complicated purification process, lowproductivity, etc. Accordingly, the purpose of the invention is toprovide a process for efficiently producing hGH using a host-vectorsystem which allows an efficient production of hGH.

Udaka et al. seeked to develop host-vector systems for the production ofheterogeneous proteins, found that Bacillus brevis can be advantageouslyused as a host, and proposed the use thereof for production of severalheterogeneous proteins (for example, Japanese Unexamined PatentPublication (Kokai) No. 63-56,277). On the other hand, the presentinventors extensively investigated the creation of recombinant plasmidscapable of efficiently expressing DNA coding for hGH using theabove-mentioned hosts, and found that the use of a promoter derived fromBacillus brevis which is to be used as a host, and of DNA necessary forsecretion in a particular manner is effective for the expression of hGH.

Accordingly, the above-mentioned purpose is achieved by the use of arecombinant DNA wherein a DNA coding for hGH is linked to the3'-terminal of a DNA containing a promoter region derived from Bacillusbrevis. Namely, according to the present invention, provided are theabove-mentioned recombinant DNA, Bacillus brevis carrying therecombinant DNA, and a process for production of hGH characterized byculturing said Bacillus brevis in a nutrient medium and recovering thehGH from the culture.

BRIEF DESCRIPTION OF DRAWING

FIG. 1 represents the result of Western blotting after submitting aculture supernatant obtained in Example 2 and a standard hGH onSDS-polyacrylamide gel electrophoresis.

FIG. 2 represents the result of a reversed phase HPLC for a purified hGHproduct obtained in Example 4 (the abscissa axis shows retention time(min.), and the symbol arrow, shows the elution time (min.) of astandard hGH).

FIG. 3 represents the result of comparison between SDS-polyacrylamidegel electrophoresis patterns in reductive and non-reductive conditionsfor a purified hGH product obtained in Example 4 and a standard hGH.

FIG. 4 is a graph comparing a purified hGH preparation obtained inExample 4 and a standard preparation for their biological activities fordifferentiation of 3T3-L1 cells to fat cells.

FIG. 5 represents a signal sequence of MWP of Bacillus brevis 47 (shownas MWP), and a signal sequence prepared by improvement of the MWP (theimproved signal sequence is shown as R2L4).

FIG. 6 represents nucleotide sequences of oligonucleotide primers usedfor site-specific mutagenesis of DNA coding for the signal sequence ofMWP.

DNA coding for hGH used in the present invention is either DNA directlyisolated from the human pituitary gland, or DNA chemical synthesized onthe basis of known amino acid sequence of hGH (Ikehara et al., Proc.Natl. Acad. Sci. USA, 81, 5956 (1984)). Any promoter functional inBacillus brevis (hereinafter, sometimes abbreviated as B. brevis) andderived from any strain belonging to B. brevis can be used. For example,as a specific embodiment, a promoter of major extracellular protein geneof B. brevis 47 (FERM-7224) or B. brevis HPD31) (FERM BP-1087) can beused. It is essential that the DNA containing a promoter region furthercontains an SD sequence, a translation initiation codon, etc., inaddition to the promoter, and further may contain a part of the majorextracellular protein gene.

To join a DNA coding for hGH to the 3'-terminal of the DNA containingsaid promoter regions, a DNA coding for hGH may be joined to the3'-terminal of DNA excised from the chromosome of B. brevis (T. Adachiet al., J. Bacteriol., 171, 1010-1016 (1989)).

Although hGH can be intracellularly or extracellularly accumulated, ifextracellular accumulation is intended, a region coding for a signalpeptide must be included in a 3'-terminal side of a DNA containing apromoter region. As a signal peptide, for example, a signal peptide of amajor extracellular protein of B. brevis 47 or B. brevis HPD31 may bementioned. A specific example of signal peptide is that of MWP (MiddleWall Protein) of B. brevis 47.

An expression vector which contains the promoter region and is used forexpression of an hGH gene is exemplified by pHY500 or pNU200 (JapaneseUnexamined Patent Publication (Kokai) No. 2-31,682, Shigezo Udaka,Nippon Nogei Kagaku Kaishi, 61, 669 (1987)).

A method for construction of an expression plasmid for hGH is well knownin the art and described, for example in Molecular Cloning 2nd. Ed., ALaboratory Manual (Cold Spring Harbor Laboratory (1990)), and thereforea desired recombinant DNA (expression plasmid for hGH) can beconstructed according to the abovementioned procedure. As a preferableexpression plasmid for hGH, pNU200-GH, etc., as prepared in the examplesdescribed hereinafter can be mentioned. A host useful for constructionof the plasmids may be any microorganism belonging to E. coli., Bacillussubtilis or Bacillus brevis, and for example, E. coli HB101, E. coliJM109, Bacillus subtilis RM141 (J. Bacteriol., 158, 1054 (1984)), B.brevis 47 (FERMP-7224), etc., may be mentioned.

A host for expression of said gene may be any host which does notprovide a strong negative influence on hGH produced by the expression ofsaid expression plasmids, and may be B. brevis 47 (FERM P-7224), B.brevis 47-5 (FERM BP-1664), B. brevis HPD31 (FERM BP-1087) or the like.Especially preferable is B. brevis 31-OK (FERM BP-4573) obtained fromthe B. brevis HPD31 by mutagenesis. Since this mutant substantially doesnot exhibit protease activity on hGH, it can stably maintain hGHproduced and accumulated in a culture (especially extracellularly).

The above-mentioned expression plasmid for hGH may be used to transforma host B. brevis according to the Takahashi's method (J. Bacteriol.,156, 1130 (1983)) or by electroporation (H. Takagi et al., Agric. Biol.Chem., 53, 3099-3100 (1989)).

A transformant thus constructed is cultured in a nutrient medium so asto produce a large amount of hGH, a major portion of which isextracellularly secreted. When the B. brevis 31-OK is used as a host,since hGH extracellularly secreted is stably maintained, the hGH can beefficiently recovered from the culture.

A nutrient medium used for culturing contains a carbon source, nitrogensource, and if necessary inorganic salts. In addition, culturing can becarried out using a synthetic medium comprising mainly a sugar andinorganic salts. When an auxotropic strain is used, a correspondingnutrient factor necessary for the growth is preferably added to themedium. In addition, if necessary, an antibiotic or antifoam is added tothe medium. Regarding culturing conditions, an initial pH of the mediumis adjusted to 5.0 to 9.0, preferably 6.5 to 7.5. Culturing temperatureis usually 15° C., to 42° C., and preferably 24° to 37° C., andculturing time is usually 16 to 360 hours, and preferably 24 to 144hours.

After finishing the culturing, to recover hGH from the culture, asupernatant and microbial cells are separated by, for example,centrifugation or filtration. Intracellularly produced hGH is extractedby disrupting the microbial cells according to a conventional method inthe art, such as an ultrasonic treatment, French press method, etc., oroptionally with further addition of surfactant. Next, hGH contained in aculture supernatant or a cell-free extract thus obtained is purifiedaccording to a conventional procedure for purification of protein, forexample, salting out, isoelectric point precipitation, variouschromatographies such as gel filtration chromatography, ion exchangechromatography or reversed phase chromatography or the like to obtaindesired hGH.

The resulting hGH can be quantitated by an enzyme immunoassay using ananti-hGH antibody, or HPLC. In addition, hGH can be quantitated from3T3-L1 cells differentiation activity to fat cells (Techniques fortissue culturing (see. Ed. 2, Nippon Soshiki Baiyo Gakukai)).

EXAMPLES

Next, the present invention is further definitely explained in theseExamples.

Example 1. Construction of human growth hormone (hGH) expression vectorand preparation of transformants

A 565 bp HinfI-SalI fragment coding for hGH was isolated from plasmidpGH-L9 (see, Ikehara et al., Proc. Natl. Acad. Sci. USA.., 81, 5956-5960(1984)). To join a signal peptide of MWP and hGH, a DNA linker waschemically synthesized. The synthetic linker and the HinfI-SalI fragmentcontaining an hGH gene were joined by T4 DNA ligase to obtain a 596 bpNcoI-SalI fragment.

On the other hand, a 27 bp NcoI-SalI fragment was deleted from a plasmidpBR-AN2 (which maintains Pst I site of ampicillin resistance gene inpBR-AN3 described in Shin Seikagaku Jikken Koza 17 Biseibutsu Jikken Ho(1 Ed.) Nippon Seikagaku Kai, p.373-377 (1992)) containing a C-terminalregion of a signal peptide of MWP (Middle Wall Protein) which is one ofthe major wall proteins of Bacillus brevis 47 (FERM p-7224), and theabove-mentioned 596 bp NcoI-SalI fragment was inserted thereinto toobtain a plasmid pBR-AN2-GH.

The plasmid pBR-AN3-GH was treated with restriction enzymes ApaLI andHindIII to isolate a 656 bp ApaLI-HindIII fragment. On the other hand,plasmid pNU200 (see, Shigezo Udaka, Nippon Nogei Kagaku Kaishi, 61,669-676(1987)) was cleaved with restriction enzymes ApaLI and HindIII toobtain a larger fragment, which was then joined to the above-mentioned656 bp AlaLI-HindIII fragment with T4 DNA ligase. The reaction mixturewas used to transform Bacillus brevis 47-5 (FERM BP-1664, IFO 14698)according to the Takahashi's method (J. Bacteriol., 156, 1130 (1983)).From the resulting erythromycin-resistant transformant, a plasmid wasisolated according to an alkaline extraction method (see, MolecularCloning 2nd ed., A Laboratory Manual, Cold Spring Harbor Laboratory(1989)), and designated pNU200-GH.

Next, the plasmid pNU200-GH was cleaved with restriction enzymes ApaLIand BclI to obtain an ApaLI-BclI fragment containing the hGH gene. Onthe other hand, plasmid pNUR2L4 containing an improved signal sequenceR2L4 (FIG. 5 (R2L4)) (SEQ ID No: 2), wherein three Leu residues had beenadded to the hydrophobic region of MWP signal sequence (FIG. 5 (MWP))(SEQ ID No: 1) and two Arg residues had been added to the positivecharge region of said MWP signal sequence, was cleaved with restrictionenzymes ApaLI and BclI to obtain an ApaLI-BclI fragment of about 3.8kbp. This DNA fragment was joined to the above-mentioned DNA fragmentcontaining an hGH with T4 DNA ligase to obtain plasmid pNUR2L4-GH asdescribed above.

According to the Takahashi's method (J. Bacteriol., 156, 1130 (1983)),the above-mentioned plasmids pNU200-GH and pNUR2L4-GH were separatelyintroduced into Bacillus brevis 31-OK (FERM BP-4573) to obtaintransformants, from which stable strains were then isolated anddesignated Bacillus brevis 31-OK (pNU200-GH) and Bacillus brevis 31-OK(pNUR2L4-GH) respectively.

Example 2. Culturing of transformants and production and secretion ofhGH

Transformants Bacillus brevis 31-OK (pNU200-GH) and Bacillus brevis31-OK (pNUR2L4-GH) obtained in Example 1, as well as control strainsBacillus brevis 31-OK (pNU200) and Bacillus brevis 31-OK (pNUR2L4) werecultured in a medium comprising polypepton 3%, yeast extract 0.2%,glucose 3%, MgSO₄.7H₂ O 0.01%, CaCl₂.7H₂ O 0.01%, MnSO₄.4H₂ O 0.001%,FeSO₄.7H₂ O 0.001%, ZnSO₄.7H₂ O 0.0001% and erhythromycin 10 mg/L (pH7.2) at 30° C. for 3 to 6 days.

The culture broth thus obtained were centrifuged to obtain supernatantswhich were then assayed for hGH concentration by an enzyme immunoassayusing an anti-hGH antibody (Table 1). Note that, the standard hGHpreparation used was purchased from Bioproducts Co. (Seikagaku KogyoK.K.).

                  TABLE 1                                                         ______________________________________                                        Production and Secretion of hGH by Transformants                                                    Days for hGH                                            Transformant          Culturing                                                                              (mg/L)                                         ______________________________________                                        Bacillus brevis 31-OK(pNU200)                                                                       6        0                                              Bacillus brevis 31-OK(pNUR2L4)                                                                      6        0                                              Bacillus brevis 31-OK(pNU200-GH)                                                                    3        6.7                                            Bacillus brevis 31-OK(pNU200-GH)                                                                    6        16.5                                           Bacillus brevis 31-OK(pNUR2L4-GH)                                                                   6        200                                            ______________________________________                                    

Next, the above-mentioned culture supernatants were subjected to anSDS-polyacrylamide gel electrophoresis according to the Laemmli's method(see, Nature, 227, 680 (1970)), and Western blotting was carried outaccording to the Burnette's method (Anal. Biochem., 112, 195 (1981))using an anti-hGH antibody. As a result, hGH was detected in the culturesupernatants of Bacillus brevis 31-OK (pNU200-GH) and Bacillus brevis31-OK (pNUR2L4-GH), and the molecular weight thereof conformed to thatof the standard preparation (FIG. 1).

Example 3- Purification of hGH

100 ml of the culture supernatant obtained in Example 2 was pretreatedby ultrafiltration (Fitron, Minisette Omega, 10k cut off), and purifiedby ion exchange chromatography (Pharmacia), reversed phasechromatography (Pharmacia) and reversed phase chromatography (Waters) soas to obtain 2.3 mg of a purified preparation of hGH.

Example 4. properties of purified hGH preparation

The properties of the purified hGH preparation obtained in Example 3were as follows:

(1) Retention time in HPLC

As a result of reverse phase HPLC, the purified hGH preparation obtainedwas substantially homogeneous (FIG. 2), and the retention time thereofconformed to that of the standard hGH preparation (a product ofBioproducts Co.).

(2) As a result by electrophoresis in 16% polyacrylamide gel containing0.1% SDS, the purified preparation behaved in the same manner as thestandard preparation both under a reducing condition and a nonreducingcondition (FIG. 3).

(3) N-terminal amino acid sequence

As a result of analysis of N-terminal amino acid sequence of thepurified preparation by pulse liquid automatic Edman degradation (Type473A Applied Biosystems), the sequence of the purified preparationconformed to that of literature (see, Bancroft, F. C., Expl. Cell Res.,79, 275-278(1973); David V. Goeddel et al., Nature, 281, 544-548(1979)), as follows:

                  TABLE 2                                                         ______________________________________                                        N-terminal amino acid sequence                                                1         2     3      4   5   6    7   8    9    10                          ______________________________________                                        Purified                                                                             Phe    Pro   Thr  Ile Pro Leu  Ser Arg  Leu  Phe                       prepara-                                                                      tion                                                                          Literature                                                                           Phe    Pro   Thr  Ile Pro Leu  Ser Arg  Leu  Phe                       ______________________________________                                    

(4) Biological activity

Biological activity of hGH was tested taking as an indication anactivity to differentiate 3T3-L1 cells to fat cells (see, Techniques fortissue culturing (2) edited by Nippon Soshiki Baiyo Gakukai, p 110-115). As a result, the purified preparation exhibited the samebiological activity as the standard preparation (FIG. 4).

Reference Example 1. Generation of Bacillus brevis 31-OK

The plasmid pNU200-GH obtained in Example 1 was introduced into a parentstrain Bacillus brevis HPD31 (Japanese Unexamined Patent Publication(Kokai) No. 63-56,277, FERMBP-1087) to obtain Bacillus brevis HPD31(pNU200GH). Cells of this transformant were diluted and suspended in T2medium, and plated on a T2 plate, which was then irradiated withultraviolet light (2J/m²). The plate was incubated at 30° C. overnight,and resulting colonies were inoculated on a 5YC medium and cultured at30° C. for 6 days. On the third day and sixth day samples of the culturewere taken and an amount of hGH contained in culture supernatant wasmeasured by enzyme immunoassay. In this case, one strain which providedan increase of the amount of hGH in culture supernatant between thethird day and the sixth day was selected and isolated. When this mutantwas compared with the parent strain, there was a remarkable differencein that the parent strain provided the decrease of the amount of hGH inculture supernatant between the third day and the sixth day, while themutant exhibited an increase of the amount of hGH after the third day.To eliminate plasmid from the mutant, the mutant was repeatedly passagedin the erhythromycin-free T2 medium, and a strain which was sensitive toerhythromycin and did not produce hGH could be easily obtained. Thisstrain was designated Bacillus brevis 31-OK.

The microbiological properties of the Bacillus brevis 31-OK(FERMBP-4573) were substantially the same as those of the parent strainBacillus brevis HPD31 (see, Japanese Unexamined Patent Publication(Kokai) No. 63-56,277), except that these strains were different in anaction to a particular heterogeneous protein. The main properties wereas follows:

Physiological properties

    ______________________________________                                        Physiological properties                                                      ______________________________________                                        Reduction of nitrate -                                                        VP test for acetoin formation                                                                      -                                                        Formation of indole  -                                                        Formation of hydrogen sulfide                                                 TSI agar medium      -                                                        Zinc acetate agar medium                                                                           +                                                        Utilization of citrate                                                                             +                                                        Utilization of inorganic nitrogen source                                      Nitrate              -                                                        Ammonium salt        +                                                        Formation of pigments (King Medium)                                                                -                                                        Urease               -                                                        Oxidase              +                                                        Acylamidase          -                                                        Catalase             +                                                        O-F test             no-decomposition                                         Decomposition of gelatin                                                                           -                                                        Formation of acid from glucose                                                                     -                                                        Decomposition of xylose                                                                            -                                                        Decomposition of lactose                                                                           -                                                        Decomposition of maltose                                                                           -                                                        Hydrolysis of arginine                                                                             -                                                        pH allowing growth   5.5 to 8.5                                               ______________________________________                                    

Morphological properties

    ______________________________________                                        Size of cell                                                                  in liquid medium:                                                                            0.9 to 1.2 × 3.9 to 5.8 μm                            on agar medium:                                                                              0.9 to 1.2 × 2.9 to 4.2 μm                            Shape of cell  rod                                                            Polymorphism of cells                                                                        no                                                             Motility       present                                                        ______________________________________                                    

Reference Example 2. preparation of plasmid pNUR2L4

(1) Construction of plasmid M13MWP

pNU200 was digested with restriction enzymes EcoRI and HindIII (TakaraShuzo), and the resulting DNA fragment of about 670 bp was inserted intoplasmid M13mp18 which had been digested with EcoRI and HindIII so as toobtain plasmid M13MWP. A nucleotide sequence from the MWP promoter tothe signal sequence is shown in FIG. 5 (MWP) (SEQ ID NO: 1). The aminoacid sequence encoded by such nucleotide sequence is also shown in FIG.5 (SEQ ID NO: 5).

(2) Construction of plasmid M13R2

Site-specific mutagenesis was carried out using a single-stranded DNA ofthe M13MWP obtained above as a template, and using a synthetic primer R2(FIG. 6) (SEQ ID NO: 3). The site-specific mutagenesis was carried outwith an Amersham kit (Oligonucleotide-directed in vitro mutagenesissystem, Amersham). Next, the nucleotide sequence of the DNA wasconfirmed, and a desired plasmid thus obtained was designated M13R2.

(3) Construction of plasmid M13L4

According to the procedure as described in Reference Example 2.(2), asingle-stranded DNA of the plasmid M134 MWP as a template and asynthetic primer L4 (FIG. 6) (SEQ ID NO: 4) were used to obtain plasmidM13L4.

(4) Construction of plasmid pNUR2L4

The M13R2 obtained in (2) was digested with EcoRI and HpaI, and a 228 bpDNA fragment thus obtained and a 108 bp DNA fragment obtained bydigesting the M13L4 obtained in (3) with HpaI and HindIII were insertedinto pNU200 which had been digested with EcoRI and HindIII so as toobtain plasmid pNUR2L4. The nucleotide encoding signal sequence (R2L4)is shown in FIG. 5 (R2L4) (SEQ ID NO: 2). The amino acid sequence of thesignal sequence (R2L4) is also shown in FIG. 5 (SEQ ID NO: 6).

INDUSTRIAL APPLICABILITY

According to the present invention, a host-vector system useful forefficient production of human growth hormone (hGH) is provided. Namely,hGH can be efficiently produced by using Bacillus brevis as a host and arecombinant DNA wherein a DNA coding for hGH is linked to the3'-terminal of a DNA containing a promoter region derived from Bacillusbrevis as an hGH expression plasmid. Especially, a mutant Bacillusbrevis substantially not exhibiting protease activity to hGH ispreferably used as a host.

Reference to deposited microorganisms according to the provision of Rule13-2 of PCT.

Name of depository authority: National Institute of Bioscience andHuman-Technology Agency of Industrial Science and Technology

Address of depository authority: 1-3, Higashi 1-chome Tsukuba-shiIbaraki-ken 305 Japan

Deposition number and deposition date

1. FERM BP-1087 Jun. 24, 1986

2. FERM P-7224 Sep. 7, 1983

3. FERM BP-1664 Jan. 20, 1988

4. FERM BP-4573 Nov. 11, 1992

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 73 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Bacillus brevis                                                 (B) STRAIN: 47 (FERM P-7224)                                                  (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..69                                                           (D) OTHER INFORMATION: /note= "Signal Sequence of MWP of                      Bacillus brevis 47"                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGAAAAAGGTCGTTAACAGTGTATTGGCTAGTGCACTCGCACTTACT48                            MetLysLysValValAsnSerValLeuAlaSerAlaLeuAlaLeuThr                              151015                                                                        GTTGCTCCCATGGCTTTCGCTGCAG73                                                   ValAlaProMetAlaPheAla                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 88 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (vi) ORIGINAL SOURCE:                                                         (B) STRAIN: Plasmid pNUR2L4                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..84                                                           (D) OTHER INFORMATION: /note= "Improved Signal Sequence                       Derived from Signal Sequence of MWV of Baillus brevis 47"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ATGAAAAAAAGAAGGGTCGTTAACAGTGTATTGCTTCTGCTAGCTAGT48                            MetLysLysArgArgValValAsnSerValLeuLeuLeuLeuAlaSer                              253035                                                                        GCACTCGCACTTACTGTTGCTCCCATGGCTTTCGCTGCAG88                                    AlaLeuAlaLeuThrValAlaProMetAlaPheAla                                          404550                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CACTGTTAACGACCCTTCTTTTTTTCATGACCTTGTGTTC40                                    (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CCGTGCACTAGCTAGCAGAAGCAATACACTGTTAACG37                                       (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetLysLysValValAsnSerValLeuAlaSerAlaLeuAlaLeuThr                              151015                                                                        ValAlaProMetAlaPheAla                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetLysLysArgArgValValAsnSerValLeuLeuLeuLeuAlaSer                              151015                                                                        AlaLeuAlaLeuThrValAlaProMetAlaPheAla                                          2025                                                                          __________________________________________________________________________

We claim:
 1. A recombinant DNA wherein a DNA coding for human growthhormone is linked to the 3'-terminal of a DNA containing a promoterregion derived from Bacillus brevis and wherein an improved signalsequence R2L4 (SEQ ID NO: 2) is contained in a 3'-terminal site of theDNA containing the promoter region.
 2. A Bacillus brevis carrying arecombinant DNA wherein a DNA coding for human growth hormone is linkedto the 3'-terminal of a DNA containing a promoter region derived fromBacillus brevis and wherein an improved signal sequence R2L4 (SEQ ID NO:2) is contained in a 3'-terminal site of the DNA containing the promoterregion.
 3. A process for the production of human growth hormonecharacterized by culturing Bacillus brevis according to claim 2, andrecovering human growth hormone from the culture.
 4. A process for theproduction of human growth hormone characterized by culturing Bacillusbrevis 31-OK (FERM BP-4573) carrying a recombinant DNA wherein a DNAcoding for human growth hormone is linked to the 3 '-terminal of a DNAcontaining a promoter region derived from Bacillus brevis and wherein asignal sequence of MWP (Middle Wall Protein) of Bacillus brevis iscontained in a 3'-terminal site of the DNA containing the promoterregion, and recovering human growth hormone from the culture.
 5. Aprocess for production of human growth hormone characterized byculturing Bacillus brevis 31-OK (FERM BP-4573) carrying a recombinantDNA wherein a DNA coding for human growth hormone is linked to the3'-terminal of a DNA containing a promoter region derived from Bacillusbrevis and wherein an improved signal sequence R2L4 (SEQ ID NO: 2) iscontained in a 3'-terminal site of the DNA containing the promoterregion, and recovering human growth hormone from the culture.
 6. Arecombinant DNA wherein a DNA coding for human growth hormone is linkedto the 3'-terminal of a DNA containing a promoter region derived fromBacillus brevis and wherein a nucleotide sequence coding for an improvedsignal sequence R2L4 (SEQ ID NO: 6) is contained in a 3'-terminal siteof the DNA containing the promoter region.
 7. A Bacillus brevis carryinga recombinant DNA wherein a DNA coding for human growth hormone islinked to the 3'-terminal of a DNA containing a promoter region derivedfrom Bacillus brevis and wherein a nucleotide sequence coding for animproved signal sequence R2L4 (SEQ ID NO: 6) is contained in a3'-terminal site of the DNA containing the promoter region.
 8. A processfor the production of human growth hormone characterized by culturingBacillus brevis according to claim 7, and recovering human growthhormone from the culture.
 9. A process for the production of humangrowth hormone characterized by culturing Bacillus brevis 31-OK (FERMBP-4573) carrying a recombinant DNA wherein a DNA coding for humangrowth hormone is linked to the 3'-terminal of a DNA containing apromoter region derived from Bacillus brevis and wherein a nucleotidesequence coding for an improved signal sequence R2L4 (SEQ ID NO: 6) iscontained in the 3'-terminal site of the DNA containing the promoterregion, and recovering human growth hormone from the culture.